The invention concerns the use of antithrombin III with a modified conformation referred to here as activated antithrombin III (IDAAT=immune defence activated antithrombin) as a pharmaceutical preparation.
Antithrombin III is an important physiological coagulation inhibitor which inhibits circulating serine proteases without requiring a prior activation.
After forming a complex the protease cleaves the arginine 393-serine 394 bond which results in a conformation change of antithrombin and in protease-inhibitor complex formation. Heparin substantially accelerates the antithrombin-protease complex formation by binding in the amino-terminal region of antithrombin III. It is assumed that glycosoaminoglycans such as heparan sulfate assume the role of heparin on the surface of the endothelium.
Antithrombin III belongs to a family of seine protease inhibitors (serpins) which has over 100 members and it is a glycoprotein. Its polypeptide chain consisting of 432 amino acids has a molecular weight of 58000. The protein contains three intramolecular disulfide bridges and four glycosylation positions. When administered in extremely high unphysiological doses, antithrombin III reduces the mortality of sepsis in animal experiments (Dickneite and Paques, 1993). However, commercial antithrombin III preparations were not able to significantly improve the mortality or morbidity of humans suffering from sepsis.
In addition to the inhibitory effect on serine proteases and in particular on thrombin, antithrombin III was also observed to increase prostacyclin synthesis in human and bovine endothelial cells (Yamauchi et al., 1989). This increase led to a suppression of leucocytes (Kainoh et al., 1990) and is impaired by heparin (Uchiba et al., 1996) which led to the conclusion that this effect of antithrombin III is mediated by its binding to heparin-like glycosaminoglycan receptors. Moreover Stangl et al 1999 described a slight increase (1.3- to 1.7-fold) in the release of endothelin-1 or big endothelin 1 from lung tissue of rats by antithrombin III.
The form of antithrombin III is changed by inflammation-mediated processes. The so-called “natural”, “hereditary” or “constitutive” immune defence is the first defence strategy against “intruders” such as bacteria, viruses, parasites etc. and is widespread in the whole animal world. An important part of this first defence is that phagocytotic cells, in particular monocytes and PMNL (neutrophilic granulocytes) and also dendritic cells, eosinophils, blood platelets and mast cells, alone or in association with other cells migrate to the site of invasion of the pathogen (chemotaxis) and in this process penetrate through epithelia and endothelium (diapedesis).
At the site of inflammation the “foreign cells/intruders” are neutralized by phagocytosis. In this process the inflammatory cells release proteases such as elastase and cathepsin G and metalloproteases and substances which oxidize lipids, proteins and peptides.
These substances include O2, superoxide, hydrogen peroxide, peroxyrutrite, OH− radicals, hypochlorous acid HOCl, Cl2 gas, chloramine. In this connection halogenation (mainly chlorination) is an important way of killing cells. In the inflamed region the pH value is decreased to below pH 4.0 by the release of lactic acid.
The defence cells also release specific proteins and peptides for defence such as bactericidal/permeability-increasing (BPI) protein from thrombocytes and granulocytes and defensins from granulocytes.
If there is a wound or other activation of hemostasis then thrombin, factor Xa and other serine proteases are formed in this process. In addition complement activation occurs (alternative path, properdin pathway) and there is an increased synthesis and release of so-called acute phase proteins such as fibrinogen, C-reactive protein, mannose-binding protein (MBP), products of so-called immediate early genes such as thrombospondin-1 and others. Activated mast cells release soluble heparin proteoglycan which can bind to antithrombin (Linstedt et al., 1992). Antithrombin III is indirectly or directly changed by these processes and acquires completely new functions.
Within the scope of the invention it was found that antithrombin III which is directly or indirectly changed by these processes acquires completely new functions.
It was also found within the scope of the present invention that antithrombin III can also be converted in vitro into this activated form especially by processes such as oxidation, treatment with urea and guanidine hydrochloride, proteolytic cleavage, heating to 60° C., lowering the pH to 4.0 or adding an ATIII peptide which contains the sequence SEAAAS (SEQ ID NO: 1). In this process a cryptic sequence of antithrombin is exposed and allows the protein to interact with proteins such as thrombospondin, vitronectin, CD36, oxLDL, αvβ5 integrin and others.
Furthermore it was found within the scope of the invention that activated antithrombin III (IDAAT) polymerizes by self association. These polymers have repetitive binding sites for the adhering proteins and immobilize them. As a result the adhering proteins acquire functions which they do not have as soluble proteins in the plasma, serum or other body fluids and consequently they can stimulate signal transduction in membrane proteins. One of the most important interaction partners for IDAAT is thromobospondin-1 (TSP-1). TSP-1 is a modular glycoprotein composed of multiple domains which is released by many cells and is incorporated into the extracellular matrix. Blood platelets in particular contain high concentrations of TSP-1 (Flicker and Kehrel, 1993) in their α-granula and release it during their activation.
This results in a more than 1000-fold increase in the local TSP-1 concentration (Flicker and Keel, 1993). Endothelial cells, smooth muscle cells, glial cells and Jeucocytes secrete TSP-1. TSP-1 is a member of the thrombospondin family which also includes TSP-2, TSP-3, TSP-4 and the cartilage oligomeric matrix protein (COMP) (Lawler et al., 1993). Several regions of TSP-1 and TSP-2 are identical and thus several functions of TSP-1 can also be carried out by TSP-2. TSP-1 and TSP-2 have the same domain structure and can be expressed as homomers and heteromers (Bornstein et al., 1991). TSP-1 is a trimeric glycoprotein with an apparent mass of 420000 Da. Its 3 subunits have a molar mass of 180000 Da in the Lämmli SDS-PAGE system (Lawler and Hynes 1986). Electron micrographs show the trimeric structure which looks like a bola with globular ends at the amino and carboxy termini of the polypeptide chains (Galvin et al., 1985). The tree chains are linked together by disulfide bridges near to the globular amino termini. Each TSP-1 subunit contains 69 cysteine residues so that each chain has at least one free SH group. TSP-1 and TSP-2 contain similar functional domains such as the N-terminal region, a pro-collagen homologous region, type 1 TSP repeats (repetitive regions), type 2 TSP repeats, type 3 calcium binding repeats and the carboxy terminal region (Bornstein et al., 1992).
The rod-shaped connecting regions of the TSP-1 chains exhibit a calcium-dependency of the structure. In the presence of Ca2+ this structure has a length of 16 to 29.1 nm and in contrast a lend of 38.3 nm after EDTA treatment (Lawler 1986).
The conformation of TSP-1 is strongly dependent on the Ca2+ concentration (Lawler et al. 1988) and on the binding of interaction partners. Thus the binding of TSP-1 to fibronectin or heparin gives it a conformation in the absence of Ca2+ which the molecule would adopt in the presence of Ca2+ (Dardik and Lahav 1999).
Immobilized TSP adsorbed to surfaces mediates the adhesion of endothelial cells, smooth muscle cells and monocytes. This adhesion depends on the Ca2+ conformation state off le TSP-1. EDTA treatment irreversibly inhibits this process (Lawler et al. 1988). The Ca2+ form, of TSP-1 enables it to bind to cells which is RGD-mediated via integrins. The binding of CD36 also changes the conformation of the TSP-1 molecule (Leung et al. 1992). TSP-1 binds to CD36 by means of a two-step mechanism. TSP-1 only binds with high affinity to CD36 in the second step by means of the cell binding site in the properdin-like type 1 repeat.
Binding to CD36 via the peptide sequence 139-155 of CD36 enables a conformation change in TSP-1 which allows high affinity binding to the sequence 93-110. This region contains the sequence of CD36 whose binding ability is regulated by phosphorylation/dephosphorylation (Thr 92) (Asch et al., 1993). Constitutively phosphorylated CD36 binds collagen, CD36 dephosphorylated by cell activation acquires the ability to bind thrombospondin. The conformation of TSP-1 regulates its functional capability.
In addition to its ability to bind to cells via integrins and to mediate cell adhesion, other properties are also regulated by the conformation of TSP such as the modulation of fibrinolysis, inhibition of elastase and cathepsin G, improvement of wound healing and promotion of the growth of neurites.
TSP-1 deficient mice develop extensive acute and chronic organized bacterial pneumonia with massive infiltration of neutrophils and macrophages between the first and fourth week of life. Diffuse alveolar hemorrhage was observed. At a later stage of the infection a thickening and curling of the epithelium of the airways occurs compared to control mice of the sane inbred strain which lave TSP-1 (Lawler et al. 1998).
These results illustrate the importance of TSP-1 for defence against infections. TSP-1 negative mice produce significantly fewer off-spring than control animals. TSP-1 knock outs exhibit a pronounced iordotic curvature of the spine. This shows the importance of TSP-1 for the development and stabilization of the skeleton. TSP-1 deficient animals had a highly significant higher number of leucocytes in particular monocytes and eosinophils in peripheral blood.
TSP-1 is a multifunctional protein. When immobilized on surfaces, it promotes the formation of plasmin (Silverstein et al. 1986) and at the same time the immobilization protects the plasmin from inactivation by the alpha2 plasmin inhibitor. The invention described here i.e. the use of IDAAT results in an immobilization of TSP-1 on cell surfaces. The urokinase plasminogen activator (uPA) and the signal chain uPA (scuPA) bind to immobilized TSP-1 and thereby remain proteolytically active. The binding to immobilized TSP protects uPA from inhibition on by the plasminogen activator inhibitor type 1 (PAI-1) (Silverstein et al., 1990). When scuPA binds to its receptor (scuPAR) a binding site is exposed which enables the binding of cell-associated TSP-1 and vitronectin (Vn) (Higazi et al., 1996). Thus immobilized TSP-1 enables proteolytic processes to also occur in a microenvironment in which no fibrin is present.
Together with plasmin, immobilized TSP-1 activates the latent transforming growth factor beta 1 (TGF-β-1) on the macrophage surface (Yehualaeshet et al., 1999).
TSP-1 also activates TGP-β on the endothelial surface (Schultz-Cherry and Murphy-Ullrich, 1993, Schultz-Cherry et al., 1994). TGF-β inhibits the proliferation of endothelial cells and acts anti-angiogenetically. Inhibition of angiogenesis by TSP-1 has been described many times (Iruela-Arispe et al., 1999, Jiminez et al., 2000). Complex formation between TSP and FGF-β1 (basic fibroblast growth factor) is also involved in this function (Murphy-Ullrich, 1993). Absence of TGF-β leads to massive disorders in the defence against infections which can lead to death (Kulkarni et al., 1993, Shull et al., 1992). The TGF-β deficient animals additional exhibited a strong autoimmune reactivity (Letterio et al., 1996) dud to its effect on MHC class II antigen expression (Geiser et al., 1993).
Since TSP-1 immobilized on cell surfaces can activate TGF-β, it would appear that TSP-1 is involved via TGF-β in the described processes of defence against infections and autoimmune reactivity (Crawford et al., 1998). Together with TGF-β, immobilized TSP-1 regulates the proliferation of natural killer cells (NK) cells (Pierson et al., 1996). The TSP-1 deficient animals also exhibit corresponding immune deficiencies although they are less pronounced. Since the activated antithrombin which is described for the first time in this invention and which binds TSP-1, can immobilize TSP on cell surfaces, it is apparent that IDAAT can indirectly influence the activation of TGF-β.
However, TSP-1 also modulates immunological defence-relevant processes by other mechanisms. Thus a large number of microorganisms such as coagulase-negative staphyloccoci (Li et al., 2000), enterococci and Porphyromonas gingivalis fimbriae Nakamura et al., 1999) adhere to immobilized TSP-1.
Erythrocytes infected with the malaria tropica pathogen adhere to immobilized TSP-1 (Roberts et al., 1985) and to the TSP-1 receptor CD36.
The parasite itself has a membrane protein which contains TSP-1 homologous regions. This protein TRAP (thrombospondin-related-anonymous (adhesive) protein) which is transported in the erythrocyte membrane enables the parasite to mediate the adhesion of infected erythrocytes to the vessel wall (Wegelnik et al., 1999, Kappe et al., 1999).
Other pathogens such as Cryptosporidium parvum or Eimeria tenella have TSP or TSP-receptor homologous domains which they use for cell adhesion (Sulaiman et al., 1999).
The HIV-1 virus uses a CD36 (TSP receptor) domain in its surface protein GP 120 to enable the HIV virus to bind to TSP as well as to CD4 on the host cells (Crombie et al., 1998). Hence purified TSP-1 can inhibit HIV-1 infections (Crombie et al., 1998).
Several complement proteins, C9, C8 alpha and C8 beta have modules with a high degree of homology to one of the repeat modules of thrombospondin (Patthy, 1988). Antistasin, properdin and F-spondin also have other domains that are homologous to TSP. F-spondin is, like thrombospondin itself, a substance which effectively improves lesions of the nervous system (U.S. Pat. No. 5,750,502).
TSP can mediate the phagocytosis of apoptotic PMNL by binding simultaneously to apoptotic neutrophilic granulocytes (PMNL) and to macrophages. The concurrent interaction of TSP with its receptors αVβ3 integrin CD36 and CD47 is responsible for this process (Savill et al., 1992). The phagocytosis of apoptotic PMNL regulates inflammatory reactions and prevents an uncontrolled overreaction. In contrast to the phagocytosis of necrotic PMNL or PMNL that have been excessively degraded, TSP-1 mediated phagocytosis of apoptotic PMNL occurs without the release of pro-inflammatory mediators (Stern at al., 1996).
Thus a timely TSP-mediated phagocytosis prevents an inflammatory overreaction. In addition the production of proinflammatory cytokines is actively inhibited by macrophages which have taken up apoptotic PMNL (Fadok et al., 1998). The cross-linking of the TSP receptor CD47 on monocytes by TSP-1 is achieved by the invention described here and additionally results in an inhibition of the release of active interleukin 12 (IL-12) (Arman et al., 1999, Demeure et al., 2000). Interleukin 12 is an important mediator of sepsis (Steinhauser et al., 1999).
The immobilization of TSP-1 on apoptotic PMNL and on monocytes is thus a good method for positively influencing persistent chronic inflammations with drugs. These diseases also include all those in which an uncontrolled inflammatory reaction represents apart of the disease itself such as various forms of reperfusion damage, rejection reactions in organ transplantations and rheumatoid diseases.
TSP not only mediates the phagocytosis of apoptotic neutrophilic granulocytes but also of senescent eosinophils (Stern et al., 1996). This shows that TSP immobilization on the cell surface which is achieved by the invention described here is also a method for pharmaceutical treating undesired proinflammatory responses in diseases that are mediated by eosinophils such as allergies, asthma, parasitic diseases, certain tumours and connective tissue diseases. This prevents the release of highly toxic substances from the eosinophils which would damage or destroy the tissue.
TSP binds chemokines such as RANTES and thus prevents the chemokine from binding to its receptor (Barnes et al., 1998). This is another way in which TSP modulates inflammatory reactions and immune defence.
A drug which influences the function of TSP by changing its conformation or promoting its immobilization on cell surfaces of immunocompetent cells, limits undesired immune responses in diseases such as, but not limited to, rheumatoid arthritis, good pasture syndrome, insulin-dependent diabetes, pemphigus, pemphigoid, primary biliary cirrhosis, colitis ulcera, lupus erythematosus, graft-versus host disease, sepsis.
Immobilization of TSP on cell surfaces leads to a cross-linking of its receptor CD47. This cross-linking of CD47 on chronic lymphatic leukemia cells (CLL cells) causes specifically the cell death of these tumour cells (Mateo et al., 1999).
A substance which results in the binding of TSP to leukemia cells would therefore be an effective drug for treating CLL which is a lethal disease against which there is still no specific effective drug.
Thrombospondin not only inhibits neoangiogenesis by means of its effect on TGF-β but also by means of immobilization and binding to and activation of CD36.
Treatment of tumours is in mice with TSP-1 leads to the inhibition of neoangiogenesis and to the apoptosis of endothelial cells (Jiminez et al., 2000).
Inhibition of tumour angiogenesis is a good method for limiting the growth of tumours by drugs (Roberts et al., 1996). Hence a substance which mediates the binding of TSP to endothelial cells in tumours acquires antiangiogenetic and thus anticarcinogenic properties.
Neoangiogenesis can also cause blindness e.g. due to diabetes mellitus (Kaplan et al., 1999, Shafiee et al., 2000), age-related macular degeneration or prematurity in infants. A substance which mediates the binding of TSP to endothelial cells could also be used as a drug to treat this neoangiogenesis.
After injury the concentration of TSP increases significantly in the tissue around the injured region. After a balloon catheterization TSP can for example be already detected 1 hour after injury on the surfaces of the cells (Watkins et al., 1990, Munjal et al., 1990). The TSP on the cell surface increases further in the following days and then also increasingly accumulates in the matrix.
As wound healing progresses TSP disappears again from the cell membranes of the injured tissue.
One of the functions of TSP in the wound is to improve wound healing (U.S. Pat. No. 5,155,038). A substance which immobilizes TSP-1 in the wound, should improve wound healing.
TSP in the wound is expressed by the tissue cells and is also released by blood platelets during their activation.
About 1% of the total platelet protein and about ¼ of the protein content of the platelet α-granula is thrombospondin-1 (Kehrel et al., 1996). Released thrombospondin stimulates collagen-induced platelet aggregation (Kehrel et al., 1988). TSP contains a sequence RFYVVMWK (SEQ ID NO: 2) at the C-terminus which activates platelets via CD47 (Chung et al., 1999 and 1997). However, soluble TSP alone does not trigger aggregation when added to the blood, a platelet suspension or platelet-rich plasma.
Whereas platelets in suspension can only bind TSP in its Ca2+ form, platelets adhere to the high as well as the low Ca2+ form of thrombospondin immobilized on the matrix.
Hence an object of the present invention was to provide a pharmaceutical preparation which can carry out the above-mentioned functions and can thus have the expected effects.